Environmental contamination is a severe problem endangering the lives of many plants and animals, including humans. Many attempts are being made to reduce contamination by either preventing escape of the contaminants into the environment, containing the contaminants, or treating the contaminants in some way to make them less harmful. However, the first step in contaminant elimination or reduction is the identification of the contaminant followed by a determination of the quantity of contamination at the contaminated site. As the contaminated site is treated, such as by contaminant removal, degradation or encapsulation, the site is monitored to determine the effectiveness of the clean-up procedure.
The current approach of collecting soil and water samples and sending them to a laboratory for chemical analysis is time-consuming, inefficient, costly, may result in inaccurate measurements, and can pose health and safety risks to workers and the community. Ideally, the degree of contamination remaining in a contaminated site during and after the clean-up procedure should be monitored by technicians at the site. To be effective in the field, contaminant monitoring methods must be simple, portable, rapid, unambiguous, able to withstand harsh environmental conditions, and should provide results that can be visualized at the test site, preferably in the absence of instrumentation. Portable gas chromatograph/mass spectrometers for sample analysis in the field have been developed. However, the costs of production, maintenance, and operation of such instruments by highly trained technicians are understandably high. Little progress on the development of inexpensive, on-site monitoring methods has been made.
The major classes of environmental contaminants to be monitored prior to, during, and after a clean-up procedure include aromatic hydrocarbons, halogenated hydrocarbons, explosives and nitroaromatics, and pesticides.
Aromatic Hydrocarbons
Aromatic hydrocarbons, including benzene, toluene, ethylbenzene and xylene (BTEX), and polyaromatic hydrocarbons or polycyclic aromatic hydrocarbons (PAH) are organic compounds containing an aromatic ring. Since 1933, when polycyclic aromatic hydrocarbons isolated from coal tar were found to be highly carcinogenic, aromatic hydrocarbons and polycyclic aromatic hydrocarbons have been some of the most widely measured groups of environmental pollutants. These compounds are ubiquitous in the environment, mainly due to the widespread manufacture, use, storage, and disposal of petroleum products. Contamination caused by the leakage of refined petroleum products from underground storage tanks has become commonplace. Even though aromatic hydrocarbons, such as benzene have been associated with leukemia for quite some time, attempts to eliminate the use of benzene and benzene derivatives in the workplace have been unsuccessful. Because of their mutagenic and carcinogenic properties, aromatic hydrocarbons are constantly being measured in a variety of environmental matrices including air, water, soil, sediment, and tissue samples. For example, total petroleum hydrocarbon (TPH) analysis is a primary regulatory tool for establishing soil remediation goals for petroleum contamination.
Current analysis for petroleum hydrocarbons is conducted by gas chromatography (GC) or infrared (IR) methods, and hydrocarbon vapor analysis. GC and IR tests require instrumentation that must either be transported to the contamination site or a sample must be conveyed to the laboratory. Analysis by hydrocarbon vapor analysis provides rapid results in the field, but can be inaccurate and may fail to detect some of the more persistent contaminants. Some immunochemical assays have been used commercially for the rapid analysis of a variety of compounds. However, the assay results have been reported to suffer from a lack of reproducibility due to variations in the composition of the petroleum products, manufacturers or lots.
The aromatic hydrocarbon contaminants most frequently found in gasoline are benzene, toluene, ethylbenzene and xylene, collectively referred to by those skilled in the art as BTEX. In addition to its presence in gasoline and diesel fuels, benzene is involved in the production of numerous commercial products such as industrial chemicals, dyes, inks, paints, oils, plastics, rubber, detergents, explosives, and pharmaceutical drugs. Exemplary polycyclic aromatic hydrocarbons include compounds such as naphthalene, phenanthrene, benzo[a]anthracene, benzo[a]pyrene, acenaphthylene, anthracene, chrysene, dibenzo[a,h]anthracene, acenaphthene, fluoranthene, benzo[b]fluoranthene, benzo[g,h,i]berylene, fluorene, pyrene, benzo[k]fluoranthene, and indeno[1,2,3-cd]pyrene.
Halogenated Hydrocarbons
Halogenated hydrocarbons, such as the polychlorinated biphenyls (PCBs), polychlorinated naphthalenes, polychlorinated benzenes, polychlorinated phenols, polychlorinated terphenyls, polybrominated biphenyls, and chlorinated phenols, anilines, and benzenes, have been identified as common pollutants in the United States. Halogenated hydrocarbons have been and still are widely used in many industries as cleaning solvents, refrigerants, fumigants, and starting materials for the syntheses of other chemicals. This class of contaminants includes volatile halogenated hydrocarbons, such as trichloroethylene (a general solvent and degreaser and the most prevalent halogenated hydrocarbon contaminant), perchloroethylene (dry cleaning fluid), dichloroethylene, dichloroethane, dichloromethane, carbon tetrachloride, chloroform, chlorobenzene, hexachlorobenzene, pentachlorophenol (a toxic substance used as a fungicide, bactericide, algicide, herbicide and wood preservative treatment), dioxins, and dibenzofurans. Because of the extensive use and stability of halogenated hydrocarbons, hundreds of contaminated groundwater and landfill sites exist in the United States.
Polychlorinated biphenyls (PCBs) are a class of 209 discrete halogenated hydrocarbons, referred to as congeners, in which one to ten chlorine atoms are attached to biphenyl. PCBs have been used as insulating materials, dielectric fluids in capacitors and transformers, thermal conductors, fire retardants, hydraulic oils, plasticizers, printing inks, paint additives, dedusting agents, for moisture reduction and vapor suppression, and have been incorporated into pesticides and insecticides to prolong and increase toxicity. They are heat stable, non-volatile and non-biodegradable, having a half-life of approximately 25 years. Due to their widespread use and chemical and physical stability, PCBs now contaminate throughout the world, including the oceans, fresh water and estuaries.
PCBs are lipophilic, persistent and tend to bioaccumulate. The degree of lipophilicity increases with increasing ring chlorination. Generally, PCB levels increase through the food chain, with extremely high levels reported in birds, particularly fish-eating birds. PCBs have been detected in the adipose tissue, plasma and milk of humans. Occupational exposure to PCBs has been known since the 1930s to produce toxic effects. PCB-contaminated rice oil in Japan was reportedly the cause of widespread medical disorders including chloracne, pigmentation and low birth weights. PCBs have been reported to have carcinogenic or mutagenic effects on mammals, fish and birds and are therefore highly regulated by the U.S. Environmental Protection Agency (EPA). Antibodies to PCB's have been generated as described in U.S. Pat. No. 4,456,691 to Stark and various immunoassays for detecting PCBs have been reported as described in U.S. Pat. No. 5,145,790 to Mattingly et al. However, these immunoassay methods employ radioactive isotopes or other detectable moieties, such as fluorescence, that require the use of non-portable laboratory equipment, preventing assay measurements in the field.
In addition to PCBs, halogenated aromatic hydrocarbons, such as the polychlorinated or polybrominated dibenzo-p-dioxins, dibenzofurans, and diphenylethers are prevalent in the environment and waste dump sites. These compounds are lipophilic, stable, resistant to breakdown by acids, bases, heat and hydrolysis, and often extremely toxic. For example, the most toxic man-made chemical known is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, commonly referred to as dioxin). Dioxin has an acute oral LD.sub.50 of less than 1 .mu.g/kg in the guinea pig. Human exposure to dioxin has occurred inadvertently through the use of 2,4,5-trichlorophenoxyacetic acid-contaminated waste oil and through industrial accidents. The 1,2,3,7,8-pentachloro, 1,2,3,6,7,8-hexachloro, and 1,2,3,7,8,9-hexachloro isomers have toxicities comparable to that of dioxin.
Dioxin is an unwanted byproduct of many chemical processes. Although of no practical use, the extreme toxicity of dioxin makes it one of the most important chemicals known. Because of its acute toxicity, dioxin is strictly regulated by the EPA. Detection of dioxin levels as low as 1 part per trillion in soil, air, or water may justify decontamination and clean-up. For example, the discovery of dioxin at Love Canal, N.Y. resulted in homeowner evacuation, land quarantine, and a massive, expensive, and time-consuming decontamination process.
Due to its toxicity at such low levels, very sensitive analytical methods of detecting dioxin are essential. A highly sensitive method currently available requires extensive sample extraction followed by gas chromatography and high resolution mass spectrometry (GC-MS). However, this method is costly, time consuming, and labor intensive. Another method involves dioxin-induced aryl hydrocarbon hydroxylase activity in a rat hepatoma cell line. However, this method employs a heat-labile enzyme, lacks specificity, and often cannot distinguish dioxin from less toxic isomers or many other polychlorinated organic compounds. Radioimmunoassays have also been used for dioxin detection as described in European Patent Application No. 0,258,006. However, these assays require the use and detection of radioisotopes, which is not feasible in the field.
The high toxicity of dibenzofurans such as 2,3,7,8-tetrachlorodibenzofuran (TCDF) is well established. TCDF has been identified as a contaminant in PCB-containing compositions and has an acute oral LD.sub.50 of 5-10 .mu.g/kg body weight in the guinea pig. Therefore, very sensitive and specific analytical methods for detecting dibenzofurans is greatly needed. Presently available methods include radioimmunoassay, as described by Luster et al. Anal. Chem. 52:1497-1500 (1980), and column chromatography followed by high-resolution gas chromatography (GC) and quantitation by mass spectrometry (MS). Although highly sensitive, these methods require sophisticated equipment that cannot be employed in the field.
Explosives and Nitroaromatics
Vast quantities of nitroaromatic compounds are currently manufactured in the United States and abroad. The toxicity, mutagenicity, and carcinogenicity of nitroaromatics such as 2,4-dinitrotoluene are well established. The manufacture and widespread use of nitroaromatic explosives by both civilians and the military has caused extensive environmental contamination. The nitroaromatics most frequently found as environmental contaminants include 2,4-dinitrotoluene and 2,6-dinitrotoluene, which are used in plastics, dyes and munitions production; nitrophenols, which are used in pesticides; and 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitrobenzene, which are munitions wastes. TNT, a particularly persistent contaminant, is stable on soil surfaces in the environment for as many as 40 years. In addition, the explosive cyclonite (hexahydro-1,3,5-trinitro-1,3,5-triazine, commonly referred to as RDX) is a common contaminant known to be highly toxic. Therefore, the need for extensive monitoring of nitroaromatics and other explosives in the environment clearly exists.
Enzyme-linked immunosorbent assays (ELISAs) are currently available for the detection of trinitrotoluene and some other nitroaromatic compounds as described in the text IMMUNOCHEMICAL METHOD FOR ENVIRONMENTAL ANALYSIS, American Chemical Society, Columbus, Ohio, pp. 79-94, 1990. A major disadvantage to these assays are that they employ temperature sensitive components such as enzymes and therefore often fail to provide reproducible results when performed outside of the laboratory.
Pesticides
A large number of pesticide-contaminated sites exist throughout the world posing both human and ecological risks. These sites have occurred as a result of industrial spills, agricultural applications, and environmental transport. Many of the pesticides contaminating these sites are known to be extremely toxic and persistent in the environment. The most environmentally persistent pesticides are the chlorinated aliphatics such as toxaphene, DDT, chlordane, lindane, heptachlor, endrin, dieldrin, aldrin, and methoxychlor. Many superfund sites throughout the United States have been identified as contaminated with these and similar pesticides. No rapid environmental transformation pathways exist for many of these compounds, resulting in a lack of natural attenuation. For example, toxaphene is so long-lived in the environment that it has been suggested that it could outlive mankind.
Classes of pesticides that are somewhat less persistent still pose pollution problems. For example, organophosphates and organophosphorothioates, while not as persistent as the halogenated hydrocarbons, are more widely used and contaminate many sites. These classes of pesticides include methyl parathion, chlorpyriphos, fenthion and malathion.
Immunoassays
Various approaches have been described for carrying out immunoassays, which rely on the binding of analyte by an analyte receptor or antibody to determine the concentrations of analyte in a sample. Analyte-antibody assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize antibodies in substantial excess over the concentration of analyte to be determined in the assay. The early enzyme linked immunosorbent assay (ELISA) methods were "competitive" assays in which an enzyme-labeled antigen or antibody competed with an antigen or antibody to be detected for a reaction site on a bead, pad or surface to which one member of an immunologically-coupling pair was attached. Subsequently, the "sandwich" assay, a non-competitive assay, was developed. In the sandwich assay, the antibody or antigen to be determined was "sandwiched" by an immunochemical reaction between a solid surface treated with an immunological species reactive with the species to be determined and the same or a different reactive immunological species which had been coupled to a signal-generating label.
Competitive assays generally involve a sample suspected of containing analyte, an analyte analog-enzyme conjugate, and the competition of these species for a limited number of binding sites provided by the antibody. Due to competition between unbound analyte and analyte analog-enzyme conjugate for analyte-receptor binding sites, as the analyte concentration increases, the amount of unbound analyte analog-enzyme conjugate increases, thereby increasing the observed signal. The product of the enzyme reaction may then be measured using an instrument such as a spectrophotometer.
Environmental Immunoassays
It is often necessary to test samples at a site of contamination. For example, during a remediation effort involving large, expensive earth moving equipment, it is important to know whether all of the contaminated soil has been removed from a site before equipment and personnel are moved to the next job site. Immediate results regarding the extent of remaining contamination allow these decisions to be made in the most cost effective manner. Environmental conditions on such sites vary widely. It is important that a field test be rugged enough to withstand the field conditions, simple enough to be executed on site, and provide results that are reliable enough to make critical economic and regulatory decisions.
Immunoassay methods such as radioimmunoassays and ELISAs have been available for the detection of environmental contaminants for several years. The disadvantages of radioimmunoassay methods are that they utilize radioisotopes, which are known to be difficult and expensive to dispose of and require complex instrumentation for a proper interpretation of the results. The disadvantages of enzyme immunoassays are that enzymes are temperature dependent, subject to interferences by components in the sample matrix, and add time, steps, materials and cost to the assay.
The use of enzymes as labels in immunoassays requires the addition of a substrate reagent in order to visualize color development. Color development by the enzyme-substrate pair requires an incubation step that is highly dependent on temperature and can range from some number of minutes to hours. The user must carefully time the reaction according to the temperature and add another reagent called a "stop" solution to terminate color development after an optimal reaction time. If the timing is incorrect, chances for error increase. Removal of the enzyme significantly decreases the influence of ambient temperature on the assay, eliminating a significant source of variability. It also reduces the number of components, reagents, and time required to execute the test.
Enzymes are susceptible to degradation and loss of activity under adverse environmental conditions. Complicated processes are required to stabilize enzymes in a way that protects them from loss of activity without the use of refrigeration or other special storage conditions. Additionally, enzymes may be effected by components within the sample matrix, which can lead to erroneous results.
Therefore, there is an on-going need for development of new methods for the detection of analytes, such as environmental contaminants, particularly assays that are highly sensitive and can be conducted at or near the site of contamination.